2024年2月22日发(作者：)
第39卷第2期2 0 18年3月西安交通大学学报（医学版）Journal of Xi&n Jiaotong University (Medical Sciences)Vol.39 No. 2Mar. 2018◊技术方法研究！硒蛋白S截短和全长重组质粒的构建与表达黄芙萌，赵莉，马芳霞，陈钊，王莉，田李芳(西安交通大学第二附属医院肾病内科，陕西西安710004)摘要：目的构建截短和全长硒蛋白S(SelS)重组体，以期在细胞系中或动物体内高表达并观察其生物学效应。
方法用基因重组技术构建S0S的截短体和全长的重组质粒，截短体基因只包含mRNA的编码序列（CDS)，全长包
括mRNA的CDS和3'非翻译区（UTR)序列，此序列中含硒代半胱氨酸的插人序列（SECIS)。将重组质粒送公司测
序并比对。用脂质体2000将硒蛋白S0S质粒转染至细胞中，24 h后观察绿色荧光蛋白的表达，流式细胞技术检测细
胞的转染效率；收集细胞用TAol法提取总RNA，将mRNA反转录为cDNA，以此为模板用PCR扩增以观察目的基
因的表达。结果测序结果显示重组序列与目的基因完全一致，质粒构建成功。显微镜下可观察到细胞高表达绿色
荧光蛋白，经流式细胞技术验证细胞的转染效率达到40%以上。PCR产物的凝胶电泳结果显示：与对照组及其他实
验组相比，只有转染了目的基因的实验组高表达截短和全长的S0S基因。结论成功构建S0S截短的和全长的重组
质粒并转染人细胞中高表达，对观察硒蛋白截短体和完整形式在细胞系或动物体内的生物学效应提供了基础。关键词：硒蛋白S;截短体；基因重组；质粒构建
中图分类号：R392
文献标志码：ADOI：10. 7652/jdyxb201802024Construction and expression of the recombinant plasmids of
truncated and normal selenoprotein S genesHUANG Fu-meng，ZHAO Li，MA Fang-xia，CHEN Zhao，WANG Li，TIAN Li-fang(Department of Nephrology，the Second Affiliated Hospital of
Xi’an Jiaotong University，Xi’an 710004，China)ABSTRACT： Objective To construct the recombinant plasmids of normal and truncated selenoprotein S genes so
as to observe their biological function
in vitro or
in vivo. Methods We constructnormal and truncated selenoprotein genes by gene recombinant technology. The gene of truncated selenoprotein was
coding domain sequence (CDS) fragment ofmRNA; the gene of normal selenoprotein wasCDS and 3’untranslated
region (including Sec insertion sequence) fragment of mRNA. We confirmed the sequence of recombinant genes by
sending them to a company for comparison. The recombinant plasmids of normal and truncated genes of SelS were
transfected into cells by Lipofectamine 2000. After 24 hours， the expression of green fluorescent protein wasobserved and transfection efficiency was detected by FACS analysis. We collected the cells to isolate the total RNA
byTRIzol method，and then cDNA was obtained bymRNA reverse transcription and amplified by PCR. Results
The sequencingresults showed that the recombinant geneswerecompletely the sameas the target genes，indicatingt ha t we cons t rue t ed t he plasmids successfully. The expression of green fluorescen t pro t ein could be observed andtransfection efficiency was detected up t o 40% by FACS analysis. PCR resultsshowed that the target selenoprotein
gene was highly expressed in the experiment al group than in control group. Conclusion The t runcat ed and normalselenopro t ein S genes were successfully cons t rue t ed and t ransfec t ed in t o cells where t hey were highly expressed. It
lays founda t i on for observing t he biological effec t of t runca t ed and normal selenopro t ein in cell line or animal body.
KEY WORDS： selenoproteinS; truncatedprotein; gene recombination; plasmidconstruction收稿日期：2017-02-28
修回日期：2017-05-01基金项目：国家自然科学基金资助（No. 81400740);西安交通大学校基金（No. X.2014084)和西安交通大学第二附属医院院基金资助[No. RC
(XM)20140]Supported by the National Natural Science Foundation of China (No. 81400740)， the Foundation of Xi'an Jiaotong University(No. Xjj2014084)，and the Foundation of the Second Affiliated Hospital of Xi'an Jiaotong University [No. RC(XM)20140]通信作者：田李芳，主治医师，博士. E-mail: tianffang0316@优先出版：kns. cnki. net/kcms/detail/61. 1399. R. 20180202. 1919. 040. html(2018-02-02)http：//yxxb. xjtu. edu. cn
